Process and compositions for differential diagnosis of the megaloblastic anemia syndromes

ABSTRACT

A METHOD AND SEVERAL COMPOSITIONS FOR THE ACCURATE AND EARLY DIFFERENTIAL DIAGNOSIS OF THE DEFICIENCY STATES CAUSING MACROCYTIC MEGALOBLASTIC ANEMIA SYNDROME IS PROVIDED IN WHICH ONE OF SEVERAL COMBINATIONS OF SPECIFIED AMINO ACIDS OR OTHER SPECIFIC PRECURSOR COMPOUNDS ARE ADMINISTERED TO THE TEST SUBJECT IN METABOLIC LOADING DOSES AND THE URINARY EXCRETION OF CERTAIN UNUSUAL METABOLIC PRODUCTS IS DETERMINED. THE METABOLIC ANOMALY IN FOLIC ACID DEFICIENCY STATES IS FORMINOGLUTAMIC ACID (FIGLU); IN COBALAMIN VITAMIN B12 DEFICIENCY STATES IT IS METHYLMALONATE (MMA). THIS DIAGMOSTIC PROCEDURE DESCRIBED HEREIN IS POSITIVE EVEN BEFORE THE CHARACTERISTIC ANEMIA IS DEVELOPED. THE RESULTS OF THE DIAGNOSTIC PROCEDURE DESCRIBED PERMITS FROM THE ANALYSIS OF ONE URINE SPECIMEN THE ACCURATE AND DEFINATIVE IDENTIFICATION OF THE PRESENCE AND SEVERITY OF A FOLIC ACID AND/OR VITAMIN B12 DEFICIENCY STATE AND THUS AFFORDS A BASIS FOR DETERMINING SPECIFICALLY THOSE TWO DEFICIENCY STATES. OTHER ASPECTS OF THE INVENTION ARE DESCRIBED IN THE SPECIFICATION.

United States Patent Int. Cl. G01n 33/16 US. Cl. 424-9 22 ClaimsABSTRACT OF THE DISCLOSURE A method and several compositions for theaccurate and early differential diagnosis of the deficiency statescausing macrocytic megaloblastic anemia syndrome is provided in whichone of several combinations of specified amino acids or other specificprecursor compounds are administered to the test subject in metabolicloading doses and the urinary excretion of certain unusual metabolicproducts is determined. The metabolic anomaly in folic acid deficiencystates is formiminoglutarnic acid (FIGLU); in cobalamin vitamin Bdeficiency states it is methylmalonate (MMa). This diagnostic proceduredescribed herein is positive even before the characteristic anemia isdeveloped. The results of the diagnostic procedure described permitsfrom the analysis of one urine specimen the accurate and definitiveidentification of the presence and severity of a folic acid and/orvitamin B deficiency state and thus affords a basis for determiningspecifically those two deficiency states. Other aspects of the inventionare described in the specification.

This invention relates to a method and compositions for differentialdiagnosis of the megaloblastic anemia syndromes. More particularly, itpertains to the specific posi tive differential diagnosis of cobalamin(vitamin B deficiency disease syndromes from folic acid deficiencydisease syndromes. Both are characterized by causing histologically andusually clinically identical megaloblastic macrocytic anemia syndromes.

My previous patent, US. Pat. 3,157,575, issued Nov. 17, 1964, describesthe administration of metabolic loading dosages of the amino acid,histidine, and the measurement of the subsequent excretion level andamount of formiminoglutamic acid (FIGLU) into the urine, to specificallyand accurately identify the presence and severity of the folic aciddeficiency syndrome. Because over 98% of the megaloblastic anemiasyndromes in humans is caused by either a deficiency of folic acid orcobalamin or a combination thereof, this method, by specificallyidentifying folic acid deficiency, permitted a partial diagnosticseparation of the syndromes associated with the megaloblastic anemias.The red blood cell and certain other histologic characteristics of thesesyndromes are morphologically identical under the microscope.

My prior method positively identified only the syndrome and anemiacaused by folic acid deficiency. The diagnosis of cobalamin deficiencycould only be made inferentially.

The term cobalamin as used herein is directed generically to the entiregroup or organocobalt compounds possessing votamin B activity andincludes the cyano and hydroxycob-alarnins as Well as other naturallyoccurring related compounds, and complexes thereof, usually withproteins.

The term folic acid as used herein is directed generically to the entiregroup of folate derivatives or pteroylglutamate compounds, including themono, di, tri, hepta and other polyglutamates of pteroylglutamic acid,reduced forms, of each of these compounds as well as derivatives such asthe formyl, methyl, methylene, methenyl and 'ice formimino, and naturalcomplexes thereof, usually with proteins.

It is of importance that a positive diagnosis of each type of deficiencyleading to the syndrome characterized in its overt form by macrocyticmegalobl-astic anemia, be established. Therapy for each deficiency stateis different and must be specific. Administration of the non-deficientof these two vitamins to a subject deficient of the other, whiletemporarily clearing the blood abnormality, may mask other symptoms ofthe deficiency state and lead to serious consequences for the patient.

For example, administration of folic acid will clear the megaloblasticanemia of pernicious (Addisonian) anemia caused by a cobalamin (vitaminB deficiency, but it does not affect or arrest the neurologicalcomplications which are part of the vitamin B deficiency un derlying theAddisonian anemia syndrome. Folic acid, in amounts sufiicient to restorenormal blood values in Addisonian pernicious anemias, does not arrestthe progression of existing neurological complications due to vitamin Bdeficiency, and will not prevent the development of neurological lesionsin patients that were heretofore free of them. Cases have been reportedin which the administration of folic acid to cobalamin deficientpatients has appeared to precipitate an explosive, progressive andsevere form of the neurological lesions of Addisonian anemia whilepresenting a picture, based on blood counts and bone marrow findings, ofhematopoietic improvement. Since the initial neurological symptoms ofthe cobalamin deficiency underlying and leading to pernicious anemia aregenerally diffuse and nonspecific, the improving or improvedhematopoietic picture can be misleading as to the true vitamindeficiency underlying the patients disorder, and through a resultingconfusion, delay the institution of the proper specific therapy to apoint where the progression of neurological lesions may lead toirreversible changes. With continued administration of folic acid to anunrecognized pernicious anemia patient, in addition to or besides theappearance and/or progression of neurological disease, the macrocyticmegaloblastic anemia, thrombocytopenia and resulting purpura orhemorrhage, and neutropenia and infection may occur or recur.

Similarly, a positive true and definitive diagnosis of folic aciddeficiency state must be made or the administration of theraputicdosages of the unneeded cobalamin (vitamin B may produce a temporaryimprovement of the anemia, if the vitamin B is in sufliciently highdosage, and the folic acid deficiency is not too severe, whilepermitting the folic acid deficiency to continue to the point wheresevere anemia or other manifestations of the folic acid deficiencystate, such as thrombocytopenia and purpura or neutropenia may appearwith resulting serious consequences for the patient. If the folic aciddeficiency is severe, no improvement may occur to vitamin B therapy evenin high dosage, permitting the folic acid deficiency to becomeprogressively more severe With a resulting Worsening anemia, exposingthe patient to possibly serious and even fatal hemorrhage from thethrombocytopenia and fulminating and intractable infection induced inpart by the resulting neutropenia of the severe folic acid deficiencystate.

Because of the previous lack of a clinically simple and convenientmethod for the positive differential diagonsis of the specificdeficiency state causing the macrocytic megaloblastic anemia syndrome,some physicians in the past have prescribed hematinics to be takenorally and containing both of these agents. This practice is not onlyexpensive but may lead to the same serious consequences in unrecognizedpernicious anemia patients or may obscure the presence of otherunderlying disease.

As a result, the US. Food and Drug Administration has interdicted andseverly restricted folic acid from vitamin preparations which areavailable to the public without a doctors prescription.

It is an object of this invention to provide a differential diagnosticmethod for identifying the persence and severity of the specific folicacid and or vitamin B deficiency state associated with the magaloblasticanemia syndromes in incipient or fully developed states.

It is an object of this invention to provide a combination of materialsfor this differential diagnostic method which simply, uniquely, quicklyand positively differentiates the folic acid from the cobalamin (vitaminB deficiency syndrome of megalobastic anemias in humans, and identifiesthe presence of either folic acid and vitamin B deficiency or both.

It is a further object of this invention to provide a method fordiagnosis of disturbances in amino acid and/ or fatty acid utilizationand metabolism which disturbances prevent the proper utlization ofcertain specific amino acids and/ or fatty acids by the body, causinganomalies in the excretion of specific identifying chemicals, saiddistrubances resulting from deficiencies of folc acid and/ or cobalamin(vitamin B Other objects and advantages of this invention will becomeapparent from the detailed description and examples which follow.

THE INVENTION This invention provides a method for the differentialdiagnosis of folic acid and vitamin B deficency in humans whichcomprises administrating loading dosages of histidine or its non-toxicsalts in combination with a precursor of methylmalonate. The loadngdosages are the amounts of each of these substances which will causesignificant increases in the amounts of FIGLU and MMa excreted by thetissues. I have found that when loading doses, of histidine and MMaprecursor are administered to subjects with folic acid and/ or vitamin Bdeficiency, FIGLU is excreted in the urine in diagnostically increasedamounts, in folic acid deficient subjects; MMa is excreted in the urinein diagnostically increased amounts in vitamn B deficent subjects.Normal individuals as well as those with other types of anemia, do notexcrete increased amounts of these substances when loading materials areadministered in the manner described in this invention. Thedetermination of the amount of these substances excreted is preferablymade upon the pooled urine collected during the 24 hour period followingthe initiation of the loading dosages.

I have further found that the MMa precursors which are diagnosticallyeffective when administered in loading dosage combination with histidineare L-valne, DL-valne, L-isoleucine, DL-isoleucine, L-threonine,DL-threonine, thymine, L-homoserine, DL-homoserine, and their nontoxiccomplexes and salts. Of these the threonine, thymine and homoserineforms are novel, in that they have never before been reported alone orin combination, as MMa precursors useful for the diagnosis of thevitamin B deficiency state in man.

I have also found unexpectedly, that the best urinary output ofsignificant amounts of FIGLU and MMa results when the loading dosagesare administered in divided amounts within the loading period, of about12 hours. Preferably the loading dosages should be administered in threeportions at 4 hour intervals.

For adults, for example, a total loading dose of L-histidine HCl'H Oshould aggregate from about to 20 gm. depending upon the individualssize and weight. The loading dosage amount of DL-valine, for example,should aggregate from about to about 60 gm. For maximum FIGLU and MMaoutputs at a particular loading dose level, these combined aggregatesshould preferably be administered, in 3 portions with a portion givenevery 4 hours. The individual portions, under such an administrationprocedure, would range from 1.7 to 6.67 gm. of L-histidine, HCI-H O and5 to gm. of DL-valine.

In addition to the novel method which provides for 4 the concurrentadministration of the histidine and the MMa precursor, this inventionalso includes compositions for the simultaneous administration of thesematerials in specified dosages and proportions.

The compositional aspects of this invention includes the discovery thatwhile the L-form of the precursor compounds are active, the D-form isnot active. However, surprisingly, when the racemates of the amino acidsare administered, an increase in MMa excretion has been found that isfrom 30 to greater than would be expected from the L-form content ofadministered loading dosage.

I have found, also, that the amount of most of the MMa precursors, whenadministered with histidine, is different, for some unexplained reason,from the amount necessary for its administration when the MMa precursoris given alone to enhance MMa output. The amount of MMa precursor in mycombination was therefore increased above the level that might beinferred from the MMa excretion induced by the single agentadministration to obtain sufficient loading to insure positivediagnosis.

Loading dosage, as the term is used herein, encompasses the aggregateamounts of histidine and MMa precursors that are concurrently orsimultaneously administered to cause the characteristic elevatedexcretion of either FIGLU or MMa in folate or vitamin B deficientindividuals. The aggregate loading dosage of histidine, for adults, inmy composition is from about 5 to about 20 gm.; and for the MMaprecursor, the aggregate loading dosage is from about 10 to about gm.The range of the aggregate dosage amounts for the specific MMaprecursors in my combination for administration to adult subjects islisted below:

Range of aggregate loading dosage of MMa Precursors used with histidine,for adults Precursor: Grams DL-valine 15-60 DL-threonine 3 0-80 L-valine10-40 L-threonine 20-60 DL-isoleucine 15-60 L-isoleucine 10-30 'Ihymine1 0-50 DL-homoserine 4010() All of the above dosages ranges provideddiagnostically significant increases of urinary MMa in vitamin Bdeficient subjects, but not in normal control subjects, folic aciddeficient or other non-vitamin B deficient patients with various typesof anemia.

Of great interest with relation to intermediary metabolism leading toMMa, in the human, is the increase produced by threonine, thymine andhomoserine. The urinary MMa enhancing effect of the latter compounds hasnot been previously shown in humans with vitamin B deficiency. On anequimolar basis thymine produces urinary MMa increases of about /2,L-threonine about A to /5 that of L-valine, and DL-hornoserine about /6to 4 that of L-valine.

Efforts to test the enhancing effect of propionate and homocysteine onurinary MMa excretion in vitamin B deficiency which have been reportedas active MMa precursors were regularly frustrated in humans by nausea,followed often by vomiting, despite the administration of thesesubstances in a variety of menstrua, dose schedules and pH levels.

In other experiments, equimolar mixtures of L-valine and L-isoleucine;L-valine and L-threonine; and L-valine, DL-isoleucine and DL-threonine,do not produce urinary MMa excretions significantly different from thosewhich might be expected from the equimolar administration of theindividual amino acids.

The relative order of the urinary MMa enhancing activity of the MMasubstances studied, in human subjects having vitamin B deficiency, butnot in folic acid deficient or control subjects, on an equimolar L-formbasis are: (1) L-valine, (2) L-isoleucine, (3) DL-valine, (4)DL-isoleucine, (5) thymine, (6) L-threonine, (7) DL- threonine, (8)L-homoserine, and (9) DL-homoserine. No significant urinary MMaexcretion occurs in vitamin B deficiency after D-valine, L-methionine,DL-serine, L-asparagine, L-aspartic acid, beta-alanine or L-histidine,when administered in approximately equimolar amounts to those of thepreviously mentioned components.

When administered in equimolar amounts, L-valine and L-isoleucineproduce a urinary MMa increase of about the same order of magnitude,although L-valine usually, gives moderately higher values. The racemicform of these amino acids, when given in a molar amount equal to that ofthe L-form amino acid, gives urinary MMa increases in the order of 3080%that of the L-form.

I have found, further, that when the level of FIGLU in the 24 hourpooled urine following administration of a member of the group FIGLUprecursors, in the loading dosage and procedure described herein, is inexcess of about 30-35 micrograms per liter and is excreted in an amountin excess of about 35 mgm. during the 24 hours, from the initiation ofthe loading procedure, such values are diagnostic for the presence offolic acid deficiency states. Further when the levels are below theseindicated amounts, such levels signify the absence of such a deficiencystate. In general, the degree of increased excretion parallels thedegree of folic acid deficiency.

Similarly when the urinary MMa in this 24 hour pooled urine sample,following initiation of loading, shows that the MMa is excreted inamounts in excess of about 30 mgm. per 24 hour period, such amountssignify a vitamin B deficiency state. Urinary excretion of less thanabout 30 mgm. Within the 24 hours after initiation of the loadingdosage, according to the method of this invention, signifies the absenceof such a deficiency state. In general, the degree of increased MMaexcretion parallels the degree of vitamin B deficiency.

The compounds necessary for administration according to the method ofthis invention are generally not too palatable in the forms that arecommonly available. Many of them, including histidine which is acidic tosome and salty to others; valine which is not too soluble and has aslightly fatty fiavor and isoleucine which causes a lumpy feeling in thestomach actually cannot be administered suspended or dissolved in waterwithout complaints from the patients. Many vehicles for theiradministration in loading dosage have been tried. I have discovered,however, an inexpensive vehicle, apple juice, which best solubilizeshistidine and most of the MMa precursors, overcomes in an unusual waythepalatability and administration problems, with the FIGLU and MMaprecursors.

The loading dosages, in divided portions, can easily be administereddissolved or suspended to 2 to 8 ozs. of apple juice. The total loadingdosage according to this invention may be administered admixed in up toa quart of this vehicle. The palatability of the apple juice permits theadministration of the loading dosages even to children and infants.

With regard to the loading dosages for infants and children, whereprompt diagnosis should be made due to the poor enzymatic reserves, Ihave found that loading doses are slightly greater than in adults whencalculated on the basis of body weight. The loading dosage for infantsand children aggregates from about 0.08 to about 0.14 gm. per pound ofbody weight for histidine and its related FIGLU precursors and fromabout 0.08 to 1.40 gm. per poundof body weight for the MMa precursors.The specific dosage for the latter is adjusted depending upon therelative activity of the material as shown in Example 2 below.

Among the histidine compounds for administration in the compositions andfor the method of this invention, I prefer L-histidine HCLH O. Thiscompound is very palatable in apple juice and the loading dosagesdissolve readily in the amounts of juice described above. The free baseof histidine is very poorly soluble.

Among the MMa precursors useful in the method of this invention areDL-valine, L-valine, DL-threonine, thymine and DL-isoleucine, in generalorder of preference. On the basis of cost and activity DL valine ispreferred. On the basis of activity alone, L-valine has the advantage.DL-threonine is a preferred compound despite its lower activity levelbecause it is very soluble and is relatively tasteless, having only aslightly sweet after taste. Thymine is relatively inexpensive and mayunder certain circumstances oifer advantages as it is not an amino acid.DL- isoleucine is included, despite its lower patient acceptance,because of its high activity level. Because of the unexplained andunforeseen increased activity in the racemic amino acid on an equivalentmolecular basis to the L-form, as MMa precursors, and because theracemates are considerably cheaper than the L-form, which until now wasthe only form of these amino acid MMa precursors shown to be active,these forms are most suitable for diagnostic use in general clinicalpractice.

The invention in its various ramifications is exemplified in theprocedure section and the examples which follow. The proceduresdescribed are useful with details being given for a novel variation fordetermining methylmalonate. The method for the detremination offormiminoglutamic acid is not set forth in detail as it is contained inthe included references.

Further, use of the MMa precursors alone, in loading dosages and mannerof administration described, and collection of urine for a period of 24hours from the initiation of the loading dosage procedure, followed bythe assay of an aliquot of such urine for its methylmalonate content,provides a specific test for the identification and determining theseverity of vitamin B deficiency according to the criteria specifiedherein and illustrated in the examples which follow.

The examples illustrate cases wherein megaloblastic anemic subjects,having given informed consent, were given loading dosages of thecomponents and the combination of this invention in order that theparameter and safety of the procedure be explored. Detailed laboratoryfindings, providing the basis for diagnosis and for recom mendingspecific formulations and proportions can be seen in the reported data.Further, experience with a general hospital and outpatient otficepopulation, wherein these tests were run on patients suspected of havingthese metabolic deficiencies, has shown the general reliability andspecific applicability of the practice of this invention.

Experience with the combination of this invention, which has beenadministered in several hundred loading dosage tests followed by assays,indicates the validity of this differential diagnostic method and itsassociated loading dosage composition. In no case, within therecommended loading dosage ranges of the various combinations describedas effective, was there any false positive result; and no false negativeresults were encountered.

While all of the assays have been performed according to the preferredprocedure, this invention is not limited to these procedures since anysufiiciently accurate method for assaying FIGLU and MMa should lead tocomparable diagnostic results.

Since all the compounds are normal components of food they are safe fororal administration. Moreover, my experience with the compounds aloneand in combination has given rise to no contraindication for their administration according to this invention. This experience includessubjects ranging from infants, pregnant women and subjects in variousstages of folic and vitamin B deficiency states.

SAMPLE COLLECTION Urine from the test subjects is collected in suitableclean containers to which sufiicient acid (usually HCl) is added to keepthe pH of the final volume below 2.0.

Under these conditions, the MMa and FIGLU in the urine is stable forweeks at room temperature. Just prior to assay, an appropriate aliquotof urine is filtered or centrifuged to remove any sediment, and the pHis adjusted appropriately for the FIGLU or MMa assay. The volume of thetotal collection of urine is measured and recorded. Preservatives may beadded if they are found not to interfere with the particular assaymethods.

ASSAY PROCEDURES While the presently preferred determination of FIGLUand MMa are performed on urine specimens, it is possible to perform suchdeterminations, by variations of the methods set forth below on bloodplasma and other body fluids where these deficiency indicator substancesappear in greater than normal concentrations after administration ofloading dosages of the precursor compounds described therein.

METHODS FOR THE ASSAY OF FIGLU Several methods for measurement of thepresence and concentration of FIGLU have been developed. These methodsfall into the following general classes:

(1) Enzymatic assays (2) Microbiological assays (3) Chromatographicassays (4) Chemical assays These assays have been outlined and describedin the following reference: Luhby and Cooperman, Advances in MetabolicDisorders, vol. 1, pp. 263 to 334, Academic Press, New York, N.Y., 1964.

With suitable modifications each of these can be used for estimating thepresence and amount of certain concentration ranges of FIGLU in urineand other body fluids.

The enzymatic assays presently available are preferred for theestimation of FIGLU in urine. One such assay is the hog liver FIGLUtransferase-cyclodeaminase enzyme (T-C) assay described in US. Pat. No.3,157,575 and in Lubby and Cooperman, Advances in Metabolic Disorders,vol. 1, pp. 263 to 334, Academic Press, New York, N.Y., 1964. Theaforementioned sources provide the details of the preparation of thereagents, handling of the unknown sample, the method for carrying outthe assay and calculating results. This is the preferred method becauseit has the greatest sensitivity of the available assays, being able todetect with confidence as little as 1 to 2 mgm. of FIGLU per ml. perurine. In addition, it is the most specific for FIGLU and is notinfluenced by in terfering compounds commonly occurring in urine,especially in those patients concerned herein. This assay has thefurther advantage of simplicity of performance, reproducibility ofresults, and lends itself to determination of FIGLU in multiplespecimens in the average clinical laboratory.

Two other enzymes methods have been described including one by Silvermanet al. (J. Natl Cancer Inst., 20, 71, 1958) which uses a microbiologicalassay for determination of the end product, and a modification of thelatter by Chanarin-Bennet (Brit. Med. 1., vol. 1, pp. 27 and 985, 1962)which converts the end product to a substance measurablespectrophotometrically.

All three of these assays are sufficiently sensitive to be used fordetecting the diagnostically important ranges of urinary FIGLUconcentrations. However, the T-C enzyme assay, as noted, is preferred.This enzyme method, set forth in detail in the aforementioned patent,was used for the determination of the FIGLU data reported in theappended clinical examples.

The microbiological assays, using Lactobacillus arabinosus as the testorganism, have good specificity but are generally too cumbersome for theroutine clinical laboratory.

A number of chromotographic assays have achieved popularity and arereadily performed with equipment commonly available in the averageclinical laboratory. However, such assays are qualitative and do notreadily detect urinary FIGLU at concentrations below to micrograms perml. In addition, interfering substances may confuse the readings.

Chemical assays for FIGLU are available. Two such have been described inthe reference cited earlier. These assays although simple to performlack suflicient specificity and sensitivity to be presently useful fordeterminating FIGLU in urine in patients with which this invention isconcerned.

METHODS FOR THE ASSAY OF METHYLMALONATE The concentration of methylmanicacid (methylmalonate) can be measured by a variety of techniques whichfall into three main classes, vapor phase chromotography, thin-layerchromatography and liquid colorimetry.

Vapor phase chromotographic techniques are the most sensitive. Thosethat are useful here involve the solvent extraction of the MMa from theurine and the direct determination of methylmalonate according to theprocedure of Hoffman and Barboriak (Anal. Biochem. 18, 10 (1967)), or bydeterminations on volatile derivatives of methylmalonate (Cox and White,Lancet 2, 353-856, 1962). Experience with these procedures indicates itis desirable to form the volatile derivatives prior to vapor phasechromatography since the native acid is decomposed in and on the columnduring the procedure. The assay procedures which are based on solventextraction of urinary MMa and the determination of its concentration byvapor phase chromotography, while they are the most sensitive andspecific assays presently available, are cumbersome, lengthy and requireboth special apparatus and highly trained personnel for the use of theapparatus and interpretation of the resultant data.

A method combining solvent extraction of the MMa from urine with thinlayer chromotography of the residue has been described (Hinterberger,B-ashir and Jones, Proc. Australian Assoc. of Clin. Biochem., 1, 143,1965). The procedure is essentially qualitative and satisfactorymodifications for the quantitative aspects of this invention have not asyet been perfected. Other methods under study involve molecular sieveschemical charges, enzyme microbiological conversions, electron capturemethods and liquid phase chromotography.

The other main group of methods which are more useful for the averageclinical laboratory are based on colorimetric assays. An excellentmethod for the determination of urinary MMa has been suggested byGiorgio and Plant (J. Lab. Clin. Med. 66, 667-676, 1965). This methodconcentrates the methylmalonate on a weakly basic anion exchange resinfrom which it is eluted and coupled with diazotized p-nitroaniline. Athigh pH, an emerald green color develops, with a molar absorbency peakof about 10,000 at 620 m wave length. Experience has shown theabsorbency to be linear over a concentration range of 0.6 to 12 ,ugm.per ml. which under the conditions suggested by the authors provides adetermination range of MMa concentrations of from 12 to 240 mgm./ literof urine.

Although the weaklybasic anion exchange resin employed above removes theMMa from the urine onto the resin, it also removes several otherdiazo-reactive substances which then appear at concentrations of from 25to 70 mgm. liter as determined at the critical MMa wave length. Thepresence of these compounds renders less precise the estimation of theurinary MMa by this method, particularly when the latter is in thediagnostically lower ranges.

In order to overcome this problem, I prefer to use a modification of theGiorgio and Plant procedure whereby a second ion exchange resin, astrongly acidic cation exchange resin in the hydrogen phase is employedto treat the eluate from the weakly basic anion exchange resin. Thisstrongly acidic cation exchange resin removes most of the interferingsubstances, while allowing the MMa to pass through thus permitting amore accurate and confident estimation of urinary MMa at levels as lowas 20 mgm./ liter of urinary sample.

A detailed description of my preferred clinical analytical procedure forMMa follows.

MATERIALS AND REAGENTS (1) Resin columns:

Resin I: A weakly basic anion exchange resin is used, such as Dowex AG 3x 4 which has polyalkylamine functional groups attached to astyrene-(4%) divinylbenzene polymer lattice, 200-400 mesh particle size,in the chloride form. The resin is washed with ten volumes of tripledistilled water and suspended in one to two volumes of distilled wateruntil used. It may be stored in a dark glass bottle at 4 C. for severalmonths.

Resin II: A strongly acidic cation exchange resin is used, such asDowex, 50 W x 8 which is Composed of nuclear sulfonic acid exchangegroups attached to a styrene-(8%) divinylbenzene polymer lattice, 200400mesh particle size, in the hydrogen form. The resin is washed and storedas above.

(2) p-Nitroaniline: A 0.075% solution is prepared by dissolving 375 mg.of recrystallized reagent grade pnitroaniline in 500 ml. of 0.2 Nhydrochloride acid. The solution may be stored in a dark glass bottleand remains stable for many months at room temperature.

Alternatively, for measurement of low concentration of methylmalonate(1-100 mg./liter), 0.025 to 0.065% p-nitroaniline solutions can be used.For concentrations between 500 and 3000 mg. of methylmalonate, a 0.01%solution may be desirable.

(3) Acetate bufier at pH 4.3: The buffer is prepared by dissolving 8.2gm. of anhydrous sodium acetate in distilled water and making thesolution up to 100 ml. with additional distilled water. The pH isadjusted to 4.3 with acetic acid.

' (4) 0.5% sodium nitrite: For lower concentrations of thep-nitroaniline reagent, 0.20 to 0.45% NaNO may be desired.

(5) 0.2 M sodium acetate.

(6) 3 N sodium hydroxide.

(7) 8 N sodium hydroxide.

(8) 0.1 N hydrochloric acid: Adjust to this pH 1.1, if necessary.

(9) Preparation of diazo reagent: 4.0 m1. of the 0.5% NaNO reagent isadded to ml. of the 0.075% pnitroaniline reagent at room temperature.The mixture is cooled in an ice bath to 4 C. and 4.0 m1. of cold 0.2 Msodium acetate reagent is added. The resulting diazo reagent is stablefor 24 hours at 4 C.

(10) Methylmalonic acid standards: Two methylmalonic acid standards areprepared, one containing 0.005 M and the other 0.01 M methylmalonicacid.

DETAILS OF ASSAY PROCEDURE Resin column preparation and treatment ofsample: A suspension of Resin 1 is placed in a 1 x cm. glass column witha sintered glass plug and packed lightly with gentle air pressure to adepth of 1 x 2.5 cm. It is preferred that the column depth of the resindoes not exceed 2.7

An appropriate sized aliquot of the test subjects urine is filtered orcentrifuged to remove sediment and the pH adjusted to 6.5 withmoderating strong alkalis.

A 5.0 ml. aliquot is allowed to flow by gravity through the column. Theresin is then washed two times with 50 ml. portions of distilled water.The methylmalonic 10 acid is then eluted from hte resin by gravity flowwith 20.0 ml. of 0.1 N HCl.

A suspension of Resin II is introduced into another 1 x 20 cm. glasscolumn and lightly packed to a depth of 4 cm. by gentle air pressure. To10 ml. of the above eluate from Resin I, is added 1.0 ml. ofconcentrated HCl, and the solution placed on the Resin H column andallowed to flow through by gravity. The efiiuent is then adjusted to pH1.1 with 8 N NaOH.

Color development: In 13 x 75 mm. glass tubes, 1.0 ml. of the acidifiedefliuent from the Resin II column above is added to 1.5 ml. of 1.0 Macetate buffer (pH 4.3). 1.5 ml. of cold diazo reagent is then added,the contents mixed and the mixture heated for 3.0 minutes in a wtaerbath at -94 C. 1.0 ml. of 3.0 N NaOH is then added to the hot mixture.The tubes are stoppered, the contents mixed, and the tubes are removedfrom the water bath and allowed to cool at room temperature for 10minutes.

For concentrations of expected urinary MMa above 240 mg./liter, anappropriately smaller amount of the acidified efiluent than 1.0 ml. isemployed; the diflerence in volume being made up with 0.1 N HCl.

The color developed is then read in a spectro photom eter as 620millimicrons employing suitable blanks and a methylmalonic acidstandard.

Each set of determinations is accompanied by a reagent blank containing1.0 ml. 0.1 N HCl, 1.5 ml. diazo reagent, 1.5 ml. acetate buffer; andtwo methylmalonic acid standards: The first MMa standard contains 0.05ml. of 0.005 M, the second standard 0.05 ml. of 0.01 M methylmalonicacid. each added to 0.95 ml. 0.1 N HCl, in separate tubes, to which 1.5ml. diazo reagent and 1.5 acetate bufier are added. These are thentreated from this point in the manner described for the sample above.

The optical density (O.D.) of the standard, the reagent blank and theunknowns are read in 1 cm. light path cuvettes in a spectrophotometer at620 millicrons.

Under standard conditions of assay, the following formula is used tocalculate the concentration of methylmalonic acid per liter of urine:

O.D. sample-OD. reagent blank O.D. standard-OD. reagent blank 1000volume eflluent urine volume effluent aliquot vol.

mg. MMa/liter urine X (standard X 1 See the following equation:

0.D. standard (corrected):

O.D. 0.005 M standard (corrected) 2+O.D. 0.1 standard (corrected) 2' mg.MMa/liter urine:

The concentration of methylmalonic acid per liter of urine in normalcontrol subjects by this method has been less than about 30-40 mg.

EXAMPLE 1 The following experiments illustrate the efiect ofmethylmalonate precursors and histidine, administered alone and invarious loading dosage combinations, upon urinary methylmalonate andformiminoglutamic acid excretion in vitamin B deficient, folic aciddeficient and normal subjects.

1 1 Subject A VITAMIN B12 DEFICIENCY SEVERE with megaloblastic anemiadue to B deficiency but not folic acid deficiency wherein the loadingdosages are given at the same time and the determination of the urinaryMMa and FIGLU content is made from a single urine collection. In theexperience with subject B below, the value of the specific diflerentialdiagnostic aspects of this invention is illustrated where the baselineendogenous 24 hour urinary MMa excretion is not diagnostic for Bdeficiency.

The tests in Experiment B illustrate the 24 hour urinary MMa augmentingaction of DL-threonine when given alone and when given in combinationwith histidine. The test of Experiment B, day 6, shows the decreased butstill considerable activity of DL-t-hreonine when given together withhistidine. The test of Experiment C, day 4, illustrates Urine Excretione/ y) Total Dose Exper Compound loading administ. MMa FI GLU Dayadministered dose, g. method total A total Experiment A 1 None 200 2.5 2DL-valine 20 6 816 616 3.0 3 None 210 2. 4 L-llistidine 15 g. x3q. 4b.".205 H 16.0 5 None 215 3. 2 6 L-histidine DL-valine +20 5g.+6.7g. x 3 q.411 440 225 16.9

Experiment B 1 None 180 2. 0 2 DL-threonine 354 174 2. 2 3 None 185 3.24 L-histidine 15 5g.x3q.4h 200 17.0 5 None 195 4. 5 6DL-thrconine+L-histidine -1-15 6.67+5 g. x 3 q. 4h 280 16. 5

Experiment 0 l None 190 3.4 2 DL-threonine 30 10 g.x3q.4h 630 440 4.6 3None 200 2.0 4 DL-threonine+L-histidine 30+15 10 g. +5 g. x 3 11.4 h22.8 5. l 6- 15-1-20 5 g.+6.7 g. x 3 q.h 1,00 200 18. 7 7 None 180 4.3L-h' tidine* thymine 15 *10 5g. *3.3x3q.4h 360 170 16. 1

Each precursor given concurrently, hr. after the other, notsimultaneously.

This patient subsequently made a classical hematological response to 3g. vitamin B intramuscularly daily for 3 days, confirming in this mannerthe presence of vitamin B deficiency (Luhby and Cooperman, adv. inMetab. Dis. 1, 263-334, 1964, Academic Press, New York, N.Y.).

In the above practice of the invention, it can be seen that in a vitaminB deficient subject, DL-valine administered alone, produced a strikingincrease in the urinary MMa excreted during the 24-hour periodcommencing with the loading dose administration; during the latterperiod the urinary FIGLU excretion was unaltered; subsequently histidinealone did not significantly affect the 24 hour urinary MMa excretion,and although the 24 hour urine collected from the commencement ofhistidine loading showed a moderate increase in urinary FIGLU output,this level is below the level which I have found is diagnostic for folicacid deficiency.

In subsequent administration of histidine and DL-valine in combination,the net 24 hour output of MMa in the urine above baseline was in theorder of /3 that when the same dose of DL-valine was given alone.Nevertheless, the increase of MMa was striking. Although in this patientthe high (about 200 mg.) baseline 24 hour urine MMa excretion isindicative of vitamin B deficiency, the test of experimental day 6illustrates how the invention opcrates as a specific differentialdiagnostic method to furthe! identify the specific B deficiency state ina subject how the reduced efiec't of giving an amino acid MMa precursorsimultaneously with histidine can be overcome and provides an example ofthe data basis for the different and increased proportional loadingdosages recommended in the combinational aspects of the invention with aFIGLU precursor, as compared to the use of the MMa precursors alone.Tests of Experiment C, day 6 and day 8, illustrate the differentialdiagnostic value of combinations of DL-isoleucine and histidine andthymine and histidine, where in each of the latter combinations produceda striking increase of the 24 hour urinary MMa output, but nosignificantly diagnostic increase of 24 hour urinary FIGLU, signifyingin each instance, the presence of vitamin B deficiency but not folicacid deficiency.

Subject B 'Female, age 36 years, lbs. body weight.

Diagnosis: Pernicious anemia, treated (early relapse).

Clinical data: No complaints Hgb. 13.2 gms. percent, RBC 3.68milli0n/mm. mean RBC corpuscular volume 98, absent gastric intrinsicfactor, serum vitamin B activity level 205 /.L,lLg-/II11., serum folicacid (L. casei) activity level 8.1 mag/ml marrow cytology equivocallymegaloblastic.

Loading dosages were administered orally, and urine collected in themanner described in text. Symbols similar to those above.

VITAMIN B12 DEFICIENCY MILD Urine excretion,

mg. 2. 7 Total loading MMa FIGLU Exper.Day. Compound administered dose,g. Dose Admirdst. Method total A total Insignficant rise.

Subject C Female, age 57 years, 115 lbs. body weight. Diagnosis: Spruemalabsorption syndrome. Clinical data: Weakness, pallor, diarrhea. Hgb.7.2 gms percent, RBC 2.2 million per/mm. mean RBC corpus- 3O cularvolume 102M, normal gastric intrinsic factor, markedly reduced xyloseabsorption, serum vitamin B intestinal absorption of xylose, serumvitamin B activity level 360 tg/ml, serum folic acid activity (L. casei)3.0 m g/ml, marrow cytology megaloblastic 4+.

Loading dosages were administered orally, and urine collected in themanner described in text. Symbols similar to those above.

FOLIO ACID DEFICIENCY, MODERATELY SEVERE Urine excretion,

Total mg./day loading Exper. dose, MMa Incre FIGLU Day Compoundadministered g. Administration method total ment 7. 2 15 5 g. x 3 q 2308. 20 6.67g.x3q.4h 6.0 None 5. 2 L-valine+L-histidine- 20+15 6.67 g.+ g.x 3 q. 4 h 220 one 5. 6 RL-isoleuoine-l-L-histidine 20+15 6.67 g.+5 g. x3 q. 4 h 234 one 4.1 L-threonine+L-histid1ne 20+15 6.67 g.+5 g. x 3 q. 4h 242 one 8. 7 Thymine+L-histidine 10+15 3.3 g.+5 g. x 3 q. 4 h 229*L-hlSlZidlIlG-HCLHzO, given throughout above.

activity level 110 tg/ml, serum folic acid (L. casei) Subject B activity2.8 mpg/ml, marrow cytology megaloblastic 50 Male, age 7 years, 1601bsbody Weight Diagnosis: Nutritional macrocytic anemia.

COMBINED VITAMIN B 2 AND FOLIO ACID DEFICIENCY; MODERATE FOLIO ACID,MILD B12 DEFICIENCY Urine excretion,

mgJday Total loading MMa Fl GLU Exper. Day. Compound administered dose,g. Dose Administ. Method total A total 1 None- 5. 2 2 Lhistidine 22 5.7Nnnp 20 6. 2 Dlrvaline 97 77 5. 9 None--- 25 4. 1 DL-va 57 32 140 on 226. 1 Dlrvaline L hbtldlfle 15 g. 5 g. x 3 q. 4 h 85 63 126 None 20 4. 7DL-threonine L-histidine-. +15 20 g. 5 g x 3 q 75 55 *L-histidineGHLHzO,given throughout above.

Loading dosages were administered orally, and urine collected in themanner described in text. Symbols similar to those above.

Subject D Male, age 47 years, 145 lbs. body weight Diagnosis:Nutritional megaloblastic anemia.

Clinical data: Weakness and pallor Hgb. 6.3 gms percent, RBC 1.9 per cumm., mean RBC corpuscular volume 106 cu. ,u, normal gastric intrinsicfactor, normal gastro- FOLIO ACID DEFICIENCY, MILD Urine excretion,mg./day Total loading MMa incre- FIGLU dose, g. Administration methodtotal ment L-histidine. HCLHZO, given throughout above.

Sub ect F Loading dosages were administered orally, and urine collectedin the manner described in text. Symbols similar Female, age 28, 120lbs., 7 months of gestation. to those above.

Diagnosis: Macrocytic anemia of pregnancy.

Clinical data: Pallor. Hgb. 8.2 g. percent, RBC 2.1 mil- EXAMPLE 3 lionper cu. mm, marrow cytology, megaloblastic 4+. Values in normalsubjects: The range of 24 hr. urinary FOLIO ACID DEFICIENCY, MODERATELYSEVERE Urine extelretion, mg. ay Total loading MMa Incre- Exper. DayCompound administered dose, (g.) Administration method total ment FIGLU1 None 5.6 2 DL-valine. 20 6.7 g. x 3 q.4 h 4. 2 3 5.7 4 12s 5 6.7 6DL-valine 118 Subject G MMa output in normal adults (20 cases), pregnantMale age months lbs women (18 cases), and infants (12 cases), withoutload- 7 i I l Diagnosis; Coeliac disfiaw mg, was 0 to 27 mgm. Afterloading with all of the MMa Clinical data: Pallor. Hgb. 7.0 g. percent,RBC 2.1 milprecursors listed throughout the examples above, alone lionmm}, marrow cytology, megaloblastic equivocal. and in the various dosesand combinations with histidine,

FOLIO ACID DEFICIENCY, MILD Urine excretion,

mg. day Total loading MMa Incre- Exper. Day Compound administered dose,(g) Administration method total ment FIGLU 1 None 8.0 2 DL-valine 3.01.0 g. x 3 11.4 h 3 4 100 5 6 DL-valine 90 including doses of MMaprecursors listed in the earlier EXAMPLE 2 table in the text, did notgive urinary MMa excretion Total fi $7355 above 30 mgm. per day, in 320loading tests. loading Administm. The range of 24 hr. urinary FIGLUexcretion in Compound (158, E- 1011 method FIGLU normal adults, (1510cases), pregnant women (680 cases), 5 2.1 infants and children (420cases), without loading, was i8 0 to 6 mgm. per days. After loading withall of the MMa 15 1. precursors listed throughout the examples above,alone lg and in various combinations with themselves, did not pro- 104:6 duce a 24 hr. urinary FIGLU output greater than 6 mgm. 2% if in 264loading tests. The various MMa precusors when Don 30 2.0 given invarious dose combinations with histidine did not fii g i8 513 produce a24 hr. urinary PjIGLU output greater than 28 alloisoleucine. mgm. perday, 1n 362 loading tests. 3 Histidine given as the preferred compound,loading Do 10 3.6 dosage and method of administration illustrated anddef f f 38 it, scribed herein in vitamin M deficient patients, has notTh Do i8 resulted in a 24 hr. urinary FIGLU output greater than 35 nt-fiiii'gsniiijjji 20 218 mgm. per day after testing over 300 suchsubjects with L-mgthiOnineHU i8 5-; pure B deficiency. Loading with thevarious MMa DIrsei r1 e:: 20 217 precursors in over 35 patients withpure folic acid defir rgg ii g %g ciency, has not resulted in anincrease of the 24 hr. L-higtidine 116111110 15 1314 urinary MMa outputover 30 mgm., in 116 loading tests. xgqfilh 0 While the invention hasbeen illustrated by the foregoing examples with specific procedures andselected llhcremhntebove baseline subjects, it will be apparent thatvarious equivalent changes and modifications may be resorted to ornecessary, in carrying out this invention without departing from thescope and spirit thereof. It will be understood that such equivalentsare within the purview of the appended claims.

I claim:

1. A method for the dilferential diagnosis of folic acid and vitamin Bdeficiencies in humans which comprises administering to the diagnosticsubjects loading dosages of:

(a) a member of the group consisting of histidine and non-toxic saltsthereof, and

(b) at least one precursor of methylmalonate selected from the groupconsisting of thymine, L-valine, DL-

valine, L-isoleucine, DL-isoleucine, L-threonine, DL-

threonine, L-homoserine and DL-homoserine, said loading dosagesaggregating, during a loading period, from about to about 20 gm. of (a)and from about to about 100 gm. of (b); collecting the urine of thediagnostic subjects during a period of about twenty-four hours from thecommencement of said loading period; and determining from said collectedurine the respective concentration levels and total amounts offormiminoglutamic acid and methylmalonate.

2. The method according to claim 1 wherein the precursors ofmethylmalonate are administered as non-toxic salts thereof.

3. The method according to claim 1 wherein the loading dosages areadministered in at least three divided portions within a twelve hourperiod.

4. The method according to claim 1 wherein the nontoxic salt ofhistidine is L-hiStidineHCLH O and the methylmalonate precursor isDL-valine.

5. The method according to claim 1 wherein in the 24 hour urine obtainedfrom the commencement of the loading period, a urinary formiminoglutamicacid level in excess of about 30-35 micrograms per ml. and in excess ofabout 35 milligrams per 24 hours identifies folic acid deficiency, aurinary formiminoglutamic acid level below said values signifies theabsence of folic acid deficiency; a urinary methylmalonate level on thesame urine in excess of about 30 milligrams per 24 hours identifiesvitamin B deficiency, a urinary methylmalonate level below said valuessignifies the absence of vitamin B deficiency.

6. The method according to claim 1 wherein the methylmalonate precursoris the racemate of an amino acid selected from the group consisting ofvaline, isoleucine, threonine, homoserine and the non-toxic saltsthereof.

7. The composition for the differential diagnosis of folic acid andvitamin B deficiences which consists of:

(a) a member of the group consisting of histidine and non-toxic saltsthereof, and

(b) at least one precursor of methylmalonate selected fiom the groupconsisting of thymine, L-valine, DL- valine, L-isoleucine,DL-isoleucine, L-threonine, DL- threonine, L-homoserine andDL-homoserine, in loading dosages for adults aggregating from about 5 toabout 20 gm. of (a) and from about 10 to about 100 gm. of (b).

8. The composition according to claim 7 wherein said precursors ofmethylmalonate are in the form of nontoxic salts thereof.

9. The composition according to claim 7 wherein said loading dosages arecontained in a liquid vehicle and in which each loading dose iscontained in about 8-32 fluid ounces of said vehicle.

10. The composition according to claim 9 wherein the concentration of(b) in said vehicle is about 0.5 to about 10 times the concentration of(a).

11. The composition according to claim 9 wherein said vehicle is applejuice.

12. The composition according to claim 7 wherein (a) isL-histidine.HCl.H O.

13. The composition according to claim 7 wherein the methylmalonateprecursor is DL-valine.

14. The composition according to claim 7 wherein the methylmalonateprecursor is DL-threonine.

15. The composition according to claim 7 wherein the methylmalonateprecursor is thymine.

16. The composition according to claim 7 wherein the amount of (a) asL-histidine hydrochloride.H O is from about 1.7 to about 6.6 gm. perunit dose and the amount of DL-valine is from about 5 gm. to about 20gm. per unit dose; three of said unit doses aggregating a loading doseas set forth in claim 7.

17. A composition according to claim 7 wherein the methylmalonateprecursor is DL-isoleucine.

18. The composition according to claim 7 wherein the methylmalonateprecursor is the racemate of an amino acid selected from the groupconsisting of valine, isoleucine, threonine and homoserine and thenon-toxic salts thereof.

19. The method for the determination of vitamin B deficiency states inhumans which comprises administrating to diagnostic subjects loadingdosages of methylmalonate precursors selected from the group consistingof thymine and the racemates of valine, isocleucine, threonine andhomoserine and the non-toxic salts and mixtures thereof, said loadingdosages aggregating, during a loading period, from about 10 to aboutgm.; collecting the urine of the diagnostic subjects during a period ofabout 24 hours from the commencement of said loading period; anddetermining from said collected urine the concentration level and totalamount of methylmalonate.

20. The method according to claim 19 wherein the loading dosages areadministered in at least 3 divided portions within a 12 hour period.

21. The method according to claim 19 wherein in the 24 hour urineobtained from the commencement of the loading period, a urinarymethylmalonate level in excess of about 30 milligrams per 24 hoursidentifies vitamin B deficiency and a urinary methylmalonate level below30 milligrams signifies the absence of vitamin B deficiency.

22. The composition for the differential diagnosis of folic acid andvitamin B deficiencies which consists of:

(a) a member of the group consisting of histidine and non-toxic saltsthereof, and

(b) at least one precursor of methylmalonate selected from the groupconsisting of thymine, L-valine, DL- valine, L-isoleucine,DL-isoleucine, L-threonine, DL- threonine, L-homoserine and DL-homoserine, and non-toxic salts thereof in loading dosages for infantsand children aggregating from about 0.08 to about 0.14 gm. per pound ofbody weight of (a) and from about 0.08 to about 1.4 gm. per pound ofbody weight of (b).

References Cited UNITED STATES PATENTS 3,157,575 11/1964 Luhby 4249 SAMROSEN, Primary Examiner Page 1 22 8? UNITED STATES PATENT OFFICECERTIFICATE OF CORRECTIGN Patent No. 3 577, 511 D t d May 4-, 197].

Inventor(s) Adrian LQOHard It: is certified that error appears in theabove-identified patent and that said Letters Patent are herebycorrected as shox-m below:

r Col. 1, line 63 change "votamin" to vitamin Col. 3, line 4, change"persence" to presence Col. 3, line 6, change "magaloblastic" tomegaloblas tic Col. 3, 'line 21, change "distrubances" to disturbancesAlso in this line, change "folc" to folic Col. 3, line 29, change"deficency" to deficiency Col. 3, line 32, change "loadng" to loadingCol. 3, line 34, change "amounts" to amounts Col. 3, line 40, change"vitamn" to vitamin Col. 3, line 41, change "deficent" to deficient Col.3, line 50, change "L- valne"and"DL- valne" to L- valine and DL- valineCol. 4, line 45, change "dosages" to dosage Col. 5, line 54, change "to"to in Col. 6, line 15, change "in" to of Col. 7, line 40, change "Lubby"to Luhby Col. 8, line 15, change "methylmanic" to methylmalonic Col. 8,line 45, a comma after "sieves" is missing. Also in line 45, "and" after"enzyme" is missing.

Col. 10, line 1, change "hte" to the Col. 10, line 15, change "wtaer" towater Col. 10, line 32, the period after "acid" should be a comma Col.10, line 38, change "millicrons" to millimicrons Col. 10, line 55, inthe formula, change "283.02" to 283.2

Col. 10, line 60, in the formula, change "0. 1" to .OlM

Col. 13, in line with Exp-er. Day 2 of the Table below line 51,

under "FIGLU total", change numeral "5. 7" to read 15.7

Col. 13, line 66, immediately below the Table, change "CHLH O" to o Pagm 2 UNITED STATES PATENT OFFICE CERTIFICATE OF CORRILC'IIUN Patent No.3 577 511 Dated May 4, 1971 Inventoflg) Adrian Leonard Luhby It iscertified that error appears in the above-identified patent and thatsaid Letters Patent are hereby corrected as shown below:

COL. 15, first Table, Exper. Day 2', under Administration Method, change"3g. to 5g. 2

Col. 15, line 16, just below the first Table, change "HCLH t0 I-ICl.H 0

Col. 15, second Table, (Subject F) Exper. Day 6, under AdministrationMethod, change "5g. x 4h" to 5g. x 3q. +h

Col. 15, third Table, (Subject G) Exper. Day 6, under AdministrationMethod, change "0.8 x 4h" to 0.8 x 3q.4h

Col. 16, line 56, change "days" to day Col. 16, line 60, change"precusors" to precursors Col. 16, line 66, change "M to B p Col. 18,line 31 (claim 19, line 5) change "isocleucine" to isoleucine Signed andsealed this 12th day of October 1971 (SEAL) Attest:

.FIETCHER JR. ROBERT GOTTSCHALK fi zifig Officer Acting Commissioner ofPatents

